3,388,113 United States Patent Office Patented June 11, 1968 2 By the use of the p-nitrocarbobenzoxy radical the 3,388,113 guanidino radical also remains protected when the carbo PROTECTION OF THE GUAND NO GROUP OF tert-butoxy radical or the p-methoxycarbobenzoxy radical, ARGENNEDURING PEPTDE SYNTHESS BY THE p-NITROCARBOBENZOXY GROUP which may also be used for the protection of the a-amino Stephan Guttmann, Alischwil, Roger Boissonnas, Bott radical, or the tert-butyl radical for the protection of the mingen, and Janos Pless, Basel, Switzerland, assignors carboxyl radical, is split off by the action of trifluoro to Sandoz Ltd. (also known as Sandoz A.G.), Basel, acetic acid, as well as when the triphenylmethyl radical Switzerland is split off from the O-amino radical with aqueous acetic No Drawing. Filled Aug. 27, 1964, Ser. No. 392,624 acid and when an ester radical is saponified by the action Clains priority, application Switzerland, Aug. 30, 1963, of an aqueous alkali. The present invention is also par 10,776/63 0 ticularly surprising because it has been established that 2 Claims. (Cl. 260-12.5) N"-p-nitrobenzyloxycarbonyl-L-arginine is not suitable for the building up of polypeptides. The protective radical of the invention is also stable ABSTRACT OF THE DESCLOSURE towards all condensation methods hitherto known for the The guanidino group of arginine, arginine-containing building up of polypeptides, e.g., the azide method, the peptides and their derivatives may be protected by reac method of the mixed anhydrides, the method of the acti tion with p-nitrocarbobenzoxy chloride. The advantage is wated esters, the dicyclohexylcarbodiimide method and that the radical p-nitrobenzyloxycarbonyl on the guani the diimidazolecarbonyl method. This great stability of the 20 bond between the guanidino and the p-nitrocarbobenzoxy dino group is stable under the conditions used to remove radical thus makes it possible to synthesize arginine con protecting groups from the cy-amino group and the car taining polypeptides in a relatively easy manner and with boxyl group during the synthesis of peptides and may be a hitherto unknown good yield. removed at the end of all the condensations by hydrogen The protective radical of the invention may be intro olysis. Examples of the synthesis of the peptides are given duced by means of ail the sufficiently reactive functional such as L-phenylalanyl-L-arginyl-L-proline and L-arginyl derivatives of p-nitrophenyl-carboxylic acid ester, the L-arginyl-L-proline. most suitable being p-nitrocarbobenzoxy chloride. For ex ample, arginine or a polypeptide containing arginine as the last member of the chain and the c-amino radical of The present invention relates to arginine containing 30 which is protected by one of the above mentioned protec peptides and to a process for the production thereof. tive radicals, is acylated with p-nitrocarbobenzoxy chlo The basic amino acid arginine is a constituent of various ride in an aqueous alkali solution. After the polypeptide natural occurring, biologically active peptides. During the has been completely built up, the p-nitrocarbobenzoxy synthesis of polypeptides which contain arginine-whether radical may be split off before, simultaneously or after they be naturally occurring or synthetically produced the other protective radicals of the peptide by catalytic the guanidino radical of the arginine radical must be pro hydrogenation, preferably on a palladium catalyst, in tected in order to avoid undesired side reactions. The nitro organic or aqueous solution. radical, the p-toluenesulfonyl and the carbobenzoxy radi The following examples describe the process of the in cal have hitherto been used as protective radicals for the vention. guanidino radical. These three radicals, however, have various disadvantages: Thus, for example, the nitro EXAMPLE 1.-Nez-CARBOBENZOXY-N (GUAN radical is only capable of being split off by catalytic hy DINO)-p-NITROCARBOBENZOXY-L-ARGINNE drogenation, a hydrogenation which is especially tedious 90 g. of No-carbobenzoxy-L-arginine are dissolved in and often incomplete in the case of higher peptides. The a mixture of 350 cc. of 1 N sodium hydroxide and 350 p-toluenesulfonyl radical is only capable of being split 4. 5 cc. of dioxane, a solution of 97 g. of p-nitrobenzyloxy off by treatment with sodium in liquid ammonia. This carbonyl chloride in 100 cc. of dioxane is slowly added method, however, cannot be used in the case of methi and adjustment to a pH value of 12 is effected by the onine containing polypeptides and also leads to cleavages addition of 4 N sodium hydroxide. After one hour 2000 in the peptide chain in the case of many other polypep cc. of water are added, filtration is effected, the filtrate tides, whereby the yield of the desired final product is 50 is extracted with ethyl ether and the aqueous phase is considerably reduced. It is furthermore not possible to use adjusted to a pH value of 2. The precipitated oil is dis the carbobenzoxy radical for the protection of both the solved in 300 cc. of methanol which contains 12 g, of guanidino radical and the ox-amino radical of the arginine sodium hydroxide, kept for one hour at 20 and con radical or of the arginine containing polypeptide since, centrated to half its volume. 600 cc. of water are added, hitherto, no method is known for the selective splitting off acidification is effected with dilute sulfuric acid and the of the carbobenzoxy radical from the a-amino radical preciptated oil is crystallized from a mixture of ethyl without simultaneously splitting it off from the guanidino acetate/dioxane (8:2). 91 g. of No-carbobenzoxy-N (gua radical of the arginine radical. Upon catalytic hydro nidino) - p - nitrocarbobenzoxy-L-arginine are obtained. genation or by treatment with hydrobromic acid in glacial Melting point 127. Io92-- 4 in dimethylformamide. acetic acid the carbobenzoxy radical is split off from the 60 guanidino as well as from the a-amino radical. EXAMPLE 2.-N (GUANIDINO)-p-NITROCARBO It has now surprisingly been found that the p-nitrocar BENZOXY-L-ARGININE DIHYDROBROMIDE bobenzoxy radical is not split off from the guanidino radical of arginine by the reaction of an anhydrous solu 5 g. of Na-carbobenzoxy-N (guanidino)-p-nitrocarbo tion of hydrobromic acid in glacial acetic acid at room 65 benzoxy-L-arginine are dissolved in 50 cc. of a 4 N solu temperature, although the same radical is capable of being tion of hydrogen bromide in glacial acetic acid, the solu easily split off from the ox-amino radical of arginine or tion is left to stand for one hour at 20, the resulting of the arginine containing polypeptide under the same hydrobromide is precipitated with ether, filtration is ef conditions. This particular acid hydrolysis, which is com fected and washing is effected with ethyl ether. After dry paratively mild and is the method now almost exclusively 70 ing, 5.2 g. of N(guanidino) - p - nitrocarbobenzoxy-L- used for the building up of biologically highly active poly arginine dihydrobromide are obtained. Melting point peptides, can thus be employed. 130 (decomposition). cD22=--8 in methanol. 3,388,113 3 4. EXAMPLE 3-Nov-CARBOBENZOXY - N (GUANI EXAMPLE 7-No. - CARBOBENZOXY - N(GUANI DINO) - p - NITROCARBOBENZOXY-L-ARGIN DINO) - p - NITROCARBOBENZOXY-L - ARGI YL-L-PROLINE METHYL ESTER NYL - N(GUANIDINO) - p - NITROCARBOBENZ 19.8 g. of No. - carbobenzoxy-N (guanidino)p-nitro OXY - L - ARGINYL - L - PROLINE METHYLES carbobenzoxy-L-arginine and 6.0 g. of L-proline methyl TER ester are dissolved in 100 cc. of acetonitrile and 100 cc. 22.4 g. of Nox - carbobenzoxy - N(guanidino) - L - of methylene chloride, cooling is effected to -10, 8.3 g. arginine and 32.5 g. of N(guanidino)-L-arginyl-proline of dicyclohexylcarbodiimide are added and shaking is methyl ester dihydrobromide are dissolved in 20 cc. of effected for 50 hours at 20° C. After filtration, evapora dimethylformamide and 150 cc. of acetonitrile, cooling is tion is effected, the residue is dissolved in ethyl acetate, O effected to -10, 14 cc. of triethylamine and 11.3 g of washing is effected with dilute acid and aqueous Sodium dicyclohexylcarbodiimide are added and shaking is ef bicarbonate solution, the organic solution is dried and fected for 3 days at 20. Filtration is effected, the filtrate evaporated. The residue is dissolved in ethyl acetate and is evaporated, the residue is dissolved in a mixture of precipitation is effeced with ethyl ether, 12 g. of dipeptide ethyl acetate and n-butanol (9:1), washing is effected ester are obtained. Melting point 90 (decomposition). 5 with dilute sulfuric acid and aqueous sodium bicarbonate a- -25 in dimethylformamide. solution, drying is effected over sodium sulfate, concen tration is effected and precipitation with ethyl ether. After EXAMPLE 4.-No. - CARBOBENZOXY - N (GUAN reprecipitating twice, filtration and drying are effected DINO) - p - NITROCARBOBENZOXY-L-ARGIN and 22 g. of tripeptide ester are obtained. Melting point YL-L-PROLINE 20 115 (decomposition). (as -20° in dimethylform 19.8 g. of Nor-carbobenzoxy-N(guanidino)-p-nitrocar amide. bobenzoxy-L-arginine and 7.8 g. of L-proline-tert-butyl ester are dissolved in 100 cc. of acetonitrile and 100 cc. EXAMPLE 8.--Na-CARBOBENZOXY - L - PHENYL of methylene chloride, cooling is effected to -10, 8.3 g. ALANYL - N(GUANIDINO) - p - NITROCARBO of dicyclohexylcarbodiimide are added, shaking is ef 25 BENZOXY - L - ARGINYL-L - PROLINE fected for 50 hours at 20, filtration and evaporation are 10 g. of No. - carbobenzoxy - L-phenylalanyi-N (guan effected, the residue is disolved in ethyl acetate, Washing idino) - p - nitrocarbobenzoxy - L-arginyl - L - proline is effected with dilute sulfuric acid and aqueous Sodium methyl ester are dissolved in 50 cc. of dioxane and 20 ml. bicarbonate solution, drying is effected over sodium sul of 1 N. sodium hydroxide, the solution is left to stand for fate, evaporation is effected, the residue is dissolved in 30 one hour at 20, 500 cc. of water and 1 cc. of glacial ethyl acetate, precipitation with ethyl ether and filtration acetic acid are added. The oily precipitate is extracted are effected. The resulting dipeptide ester is dissolved in with chloroform, drying is effected over sodium sulfate, 100 cc. of trifluoroacetic acid, the solution is left to stand concentration is effected and precipitation with ethyl for one hour at 20, evaporation is effected, ethyl ether ether. After filtering and drying, 8.6 g. of tripeptide are is added to the residue, filtration is effected, the residue obtained. Melting point 172. al.2=-34 in 95% is dissolved in 100 cc. of 1 N ammonium hydroxide, acetic acid. washing is effected with ethyl acetate, the aqueous solu tion is acidified, the oily precipitate is extracted with EXAMPLE 9-No. - CARBOBENZOXY - N(GUANI chloroform, drying and evaporation are effected. The DINO) - p - NITROCARBOBENZOXY-L - ARGI residue is dissolved in chloroform and precipitation is 40 NYL - N(GUANIDINO) - p - NITROCARBOBENZ effected with ethyl ether. After filtering and drying, 14.3 OXY - L - ARGINYL - L - PROLINE g. of No-carbobenzoxy-N(guanidino)-p-nitrocarbobenz 8.4g. of No. - carbobenzoxy - N(guanidino) - p - nitro oxy-L-arginyl-L-proline are obtained. Melting point 115 carbobenzoxy - L - arginyl - N(guanidino) - p - nitro (decomposition). cD2=-29 in 95% acetic acid. carbobenzoxy - L. - arginyl - L - proline methyl ester are dissolved in 50 cc. of dioxane, 20 cc. of 2 N sodium hy EXAMPLE 5-NCGUANIDINO) - p-NITROCARBO droxide are added, the mixture is left to stand for one BENZOXY - L - ARGINYL-L-PROLINE METHYL hour at 20, 500 cc. of water and 1 cc. of glacial acetic ESTER DEHYDROBROMIDE acid are added, extraction is effected with a mixture of 81 g, of Nox-carbobenzoxy-N (guanidino)-p-nitrocarbo chloroform/n-butanol (9:1), drying is effected over so benzoxy-L-arginyl-L-proline methyl ester are dissolved dium sulfate, concentration is effected to 100 cc., precipi in 500 cc. of a 4 N solution of hydrogen bromide in tation is effected with ethyl ether, filtration and drying are glacial acetic acid, the solution is left to stand for one effected. 8.0 g. of No. - carbobenzoxy - N(guanidino) - hour at 20, precipitation is effected with ethyl ether, fil p - nitrocarbobenzoxy - L-arginyl - N(guanidino) - p - tration is effected, the residue is dissolved in 200 cc. of nitrocarbobenzoxy - L-arginyl-L-proline are obtained. methanol and crystallization is effected by the addition Melting point 168. c.22=-27 in 95% acetic acid. of ethyl ether. After filtering, washing and drying, 76 g. of dipeptide ester dihydrobromide are obtained. Melting EXAMPLE 10.--L - ARGINYL - L - ARGINYL - L point 120 (decomposition). Io92- -33 in 95% PROLINE acetic acid. '6. g. of the protected tripeptide obtained in Example 8 60 are dissolved in 100 cc. of 66% acetic acid and hydro EXAMPLE 6.- No . CARBOBENZOXY L - PHENYL. genation is effected in the presence of 1.0 g. of 10% pal ALANYL - N (GUANIDINO) - p - NITROCARBO ladium catalyst. After 12 hours, filtration is effected, BENZOXY - L - ARGINYL-L-PROLINE METHYL evaporation is effected and the residue is crystallized from ESTER a mixture of ethanol/ethyl ether (1:1). 2.5 g. of L-argi 9.6 g. of No-carbobenzoxy-L-phenylalanine-2,4,5-tri 65 nyl-L-arginyl-L-proline are obtained. c.22s-38 chlorophenyl ester and 12.5 g. of N(guandino)-L-argin in 6 NHC. yl-L-proline methyl ester dihydrobromide are dissolved in 20 cc. of dimethylformamide, 5.6 cc. of triethylamine EXAMPLE 11-L - PHENYLALANYL-L-ARGINYL. are added and the mixture is left to stand for 16 hours L-PROLINE at 20. After the addition of 500 cc. of ethyl acetate, 6 g. of No. - carbobenzoxy - L-phenylalanyl- N(guan washing is effected with dilute sulfuric acid and aqueous idino) - p - nitrocarbobenzoxy - L-arginyl - L - proline Sodium bicarbonate solution, concentration is effected are dissolved in 100 cc. of 66% acetic acid and hydro and precipitation is effected with ethyl ether. 13.6 g. of genation is effected at normal pressure in the presence of tripeptide ester are obtained. Melting point 130 (de g. of 10% palladium catalyst. After 12 hours, filtration composition). cD22=-36 in dimethylformamide. is effected, evaporation is effected and the residue is crys 3,388,113 5 6. tallized from a mixture of ethanol/ethyl ether (1:1). 2.9 least one of the ox-amino or carboxyl groups of said g. of L - phenylalanyl - L - arginyl - L - proline are ob a-amino acids are protected during said peptide conden tained. (c) 22=-22 in 6 N HC1. sation reactions, the improvement which comprises pro What is claimed is: tecting said guanidino radical with the p-nitrobenzyloxy 1. In a process for the manufacture of peptides built carbonyl group and at least one of the c-amino or car up from c-amino acids by condensing in a series of boxyl groups of said ox-amino acids with a pro standard peptide condensation reactions, members select tective group removable in the case of the c-amino ed from the group consisting of a-amino acids, peptides protecting group by treatment with hydrogen bro built up from c-amino acids, and derivatives thereof, and mide in glacial acetic acid, trifluoroacetic acid or wherein at least one component of said cy-amino acids is aqueous acetic acid, and in the case of the carboxyl pro arginine and the guanidino radical of said arginine and at O tecting group by treatment with trifluoroacetic acid or least one of the c-amino or carboxyl groups of aqueous alkali, then selectively removing at least one of said ox-amino acids are protected during said pep said o-amino or carboxyl protecting groups by treatment tide condensation reactions, the improvement which with hydrogen bromide in glacial acetic acid, trifluoro comprises protecting said guanidino radical with the 5 acetic acid or aqueous acetic acid in the case of removal p-nitrobenzyloxycarbonyl group and at least one of the of the c-amino protecting group or trifluoroacetic acid or o-amino or carboxyl groups of said ox-amino acids with a aqueous alkali in the case of removal of the carboxyl protective group removable in the case of the c-amino protecting group, and thereafter removing the guanidino protecting group by treatment with acid, and in the case protecting group by hydrogenolysis. of the carboxyl protecting group by treatment with acid 20 or aqueous alkali, then selectively removing at least one References (Cited of said a-amino or carboxyl protecting groups by treat UNITED STATES PATENTS ment with acid in the case of removal of the ox-amino protecting group or acid or aqueous alkali in the case of 3,247, 178 4/1966 Schwyzer et al. ---- 260-112.5 removal of the carboxyl protecting group, and thereafter 25 OTHER REFERENCES removing the guanidino protecting group by hydrogen Gish et al., J. Am. Chem. Soc. 75,5872-5877 (1953). olysis. Guttmann et al., Chimia (Aarau) 18, 185 (1964). 2. In a process for the manufacture of peptides built Li et al., J. Am. Chem. Soc. 82, 5760-5762 (1960). up from a-amino acids by condensing in a series of stand ard peptide condensation reactions, members selected 30 LEWIS GOTTS, Primary Examiner. from the group consisting of a-amino acids, peptides built up from Cz-amino acids, and derivatives thereof, and ELBERT L. ROBERTS, Examiner. wherein at least one component of said ce-amino acids is M. M. KASSENOFF, Assistant Examiner. arginine and the guanidino radical of said arginine and at